Microbiological Indoor and Outdoor Air Quality in Chicken Fattening Houses

This study was conducted at one of the largest poultry companies in Kuwait during November and December 2019 to evaluate the microbiological threats of Escherichia coli (APEC), Salmonella spp., and Aspergillus fumigatus to chickens in fattening houses by counting and identifying the microorganisms by culturing and pyrosequencing analysis. During the fattening cycle, the temperature and humidity ranged between 23.6°C and 29°C and 64.1% and 87.1%, respectively. The total bacterial population and Aspergillus fumigatus measured in the indoor and outdoor air exhibited a linear relationship during the fattening cycle. The total bacterial and Aspergillus concentrations determined during the cycle ranged between 150 and 2000 CFU/m3 and 0 and 1000 CFU/m3, respectively. E. coli and Salmonella spp. concentrations determined during the cycle ranged between 1 and 220 CFU/m3 and 4 and 110 CFU/m3, respectively. Pyrosequencing analysis of the air inside the houses at the end of the cycle revealed extensive biodiversity in the microorganisms, detecting 32 bacterial genera and 14 species. The identified species belonging to the genera Corynebacterium, Haemophilus, Streptococcus, Veillonella, and Aspergillus were identified as potentially affecting human and broiler health. The emission of potentially pathogenic bacteria to the outdoor environment from chicken housing can pose a considerable risk to human health and environmental microbial pollution. This study could guide the development of integrated control devices for monitoring microbes in broiler production facilities during chicken collection for transport to slaughterhouses.


Introduction
Te accumulation of farming experience and the development of intensive and large-scale livestock production have made researchers more aware of the signifcant role that microbial aerosols play in epidemic spread, particularly respiratory diseases caused by pathogenic microbes [1,2]. In livestock and poultry, the major infectious diseases are transmitted through the air, causing great harm and losses to the livestock industry, even threatening human health and obstructing the improvement and development of animal farming production efciency. In the 1970s, researchers initiated the study of bioaerosols released during livestock and poultry farming processes [3,4]. Bioaerosols contain a complex mixture of chicken and human-derived dander, chicken feed, bedding, and viable and nonviable microbial populations [5]. Many factors, including livestock and poultry species, farming methods, farming seasons, and stages, have been shown to afect the community structure of microbial aerosols [6,7].
In poultry broiler production, exposure to bioaerosols in houses depends on the bird growth stage, as feather dandruf and feces biomass sharply increase during the fattening period. Moreover, during the fattened bird collection for transportation to the slaughterhouse, catching birds and placing them into boxes generate many supplementary bioaerosols. Tese bioaerosols can be inhaled by forklift operators while loading crates of chickens into transport. Time-based information on the quantity and microbial composition of bioaerosols is necessary to understand the relationship between these factors and adverse health symptoms in workers and animals.
Te most commonly used technique for assessing bioaerosol microbial content is culture-dependent methods. Tis technique generated data that provide a quantitative measure of culturable bacteria and a low-resolution assessment of bacterial diversity. Nevertheless, knowledge of microbial diversity is limited because the vast majority (90-99%) of naturally occurring microorganisms cannot be cultured using standard techniques [8,9]. On the other hand, real-time quantitative polymerase chain reaction (Q-PCR) is a molecular technique widely used in research areas where reproducible and accurate bacterial quantifcation is needed. Te technique ofers an attractive alternative method for quantifying the total microbial load and assessing species-specifc profles.
A great variety of microbial air concentrations in poultry houses and their surroundings are reported in the literature. In broiler houses, the reported concentrations of airborne microorganisms include up to 168,000 CFU/m 3 [10], up to 46,000 CFU/m 3 [11], and up to 220,000 CFU/m 3 [12]. Around poultry houses, the reported number of microorganisms in the air (up to 500 m away from poultry houses) ranged from 2,200 CFU/m 3 [13] to 21,000,000 CFU/m 3 [14]. Te composition of microbial air pollutant species has been studied and analyzed in detail [11,15,16]. Te primary source of microbial contamination in poultry houses is birds, followed by feed, litter, and droppings; however, microbial counts are decreased primarily by the efciency of ventilation systems [14].
Worldwide, avian colibacillosis, salmonellosis, and aspergillosis are important microbial diseases in the poultry industry. Tese diseases establish a signifcant public health problem and represent a high cost in many countries. Terefore, having a microbial air pollution database of poultry houses will assist poultry researchers and the poultry industry in reducing and eliminating avian colibacillosis, salmonellosis, and mycotoxins from poultry focks, thereby reducing the potential hazards to public health posed by these bacterial diseases. Te current database of microbial air pollution in poultry houses in Kuwait is insufcient at present. Hence, it is essential to collect and build a microbial database for poultry house air in Kuwait for the beneft of controlling broiler and human diseases. Tis study aimed to monitor the status of two of the most important airborne pathogens (Escherichia coli (APEC) and Salmonella spp.) and Aspergillus fumigatus in Kuwait's poultry houses.

Poultry Farm Studied.
Te poultry farm selected in this study represents one of the largest poultry companies in Kuwait. Tis farm is situated 50 km from the state capital of Kuwait and is located in areas reserved for the poultry industry. Te poultry farm consists of 12 × 97 m broiler houses, each of which houses 20,000 birds. Te broiler houses were decontaminated with glutaraldehyde and embedded with wood shavings prior to each fattening cycle. Te houses are ventilated using a tunnel ventilation system. Te birds are kept there until they reach 28 days of age. Te prevalence of E. coli, Salmonella spp. and Aspergillus fumigatus in the air was determined from three selected broiler houses that received one-day-old chicks (Cobb-500) from the hatchery.

Microbial Contamination
Analysis. Bioaerosols were sampled by impaction and impingement prior to the placement of the chicks and during the grow-out period. Te samples were collected weekly from areas containing ventilation fans in the three houses. Duplicate samples for E. coli and Salmonella were collected in 20.0 ml of pyrogen-free saline (0.09% NaCl) using an impinger operating at a fow rate of 12.5 L/min for 10 min. In the impaction method, samples were impacted onto XDL, MacConkey, and sorbitol MacConkey plates at a fow rate of 28.3 L/min for 10 min, using an impactor [17]. Te impinger sample solutions were concentrated to 1 ml and stored at −20°C for DNA analysis. Aspergillus fumigatus was collected with the impact method using malt extract agar supplemented with chloramphenicol (0.5%) to inhibit bacterial growth. Field and shipping blanks were collected for quality control procedures [18]. All sampling devices were operated in the morning and were placed at 0.5 m and 1.5 m above the foor. Air temperature and humidity were recorded during the sampling using a Supco DSP990 digital psychrometer (Sealed Unit Parts Co., Inc., New Jersey, USA).

Microbial Detection and Characterization by the Standard
Culture Method. Plates from the impaction procedure were incubated at 37°C for E. coli and Salmonella and at 30°C for Aspergillus fumigatus. Colonies were counted after 48 h of incubation for bacteria and after 5 days for molds; subsequently, the colony-forming units (CFUs) were determined. Te concentration of microorganisms in colonyforming units per metric cube (CFUm −3 ) was computed based on the number of colonies counted on the plates (N) as described by the following equation [19,20]: where Q is the fow rate of the sampling pump (L min −1 and the sampling time is indicated by t (min)). Isolated colonies were further confrmed for E. coli and Salmonella spp. using biochemical confrmation (Biolog Gen III Omnilog ® II Combo System). Antibiotic sensitivity for the isolated strain at diferent concentrations was performed using the standard paper disc difusion method described by the NCCLS [21]. Identifcation of flamentous fungi was carried out by microscopic examination of the culture and the fungal material mounted in lactophenol blue stain.

Microbial Contamination Analysis.
Te studies were carried out in the autumn of 2019, when the atmospheric air temperature ranged between 21.6°C and 32.2°C, and the inside temperature in the poultry houses varied from 23.6°C to 29°C. Relative air humidity ranged between 64.1% and 87.1% indoors and between 25% and 71% outdoors. Microbial contamination analysis data showed an increase in the total bacterial count inside and outside the houses' air during the fattening process ( Figure 1). Te maximum count was reached in the second week of the process, and then, the count decreased in the fourth week. Figures 2-4 clearly show the percentage increase in the microorganisms under study (E. coli, Salmonella, and Aspergillus) compared with other bacterial counts inside and outside the houses during the autumn fattening process. Figures 5-7 demonstrate the increase in E. coli, Salmonella, and Aspergillus counts during the fattening process. During the process, indoor and outdoor air showed an increase in E. coli, Salmonella, and Aspergillus counts, with the maximum count in the second week at 0.5 m. Only E. coli showed a maximum count in the third week of the process at 1.5 m, inside and outside the air.
In addition, the frst week had the highest number of Salmonella in the outdoor air. At the 1.5 m detection level, the total bacterial concentration in the fattening houses during the process ranged from 150 ± 160 to 2000 ± 600 CFU/m 3 (Table 1), which was linearly related to the outdoor air during the fattening cycle ( Figure 8). Te highest concentration was detected in the second week of the process. Te concentrations of E. coli, Salmonella, and Aspergillus during the process ranged from 1 ± 2 to 220 ± 250 CFU/m 3 , 4 to 110 ± 43 CFU/m 3 , and 0 to 610 ± 160 CFU/m 3 , respectively, with the highest concentration in the second week of the process (Table 1). Tere was a strong positive and signifcant correlation between the total number of bacteria and Aspergillus in the air inside and outside the house at 1.5 m (r � 0.995 and 0.996, respectively; P < 0.001) ( Table 2). Te ANOVA test performed on total raw data revealed signifcant diferences in the total bacterial and Aspergillus indoor contamination related to the week (Table 3). Inside the houses, at the 0.5 m detection level, the relationship between microbial concentration and the fattening cycle week was insignifcant only for total bacterial count and Salmonella (P > 0.05). However, at the 1.5 m detection level, the relationship was insignifcant only for E. coli. Additionally, outside the houses, at the 1.5 m detection level, the relationship was insignifcant only for E. coli.
Te indoor/outdoor ratio (I/O; Table 1) was calculated using the indoor and outdoor microbial counts obtained during the fattening process sampling at 1.5 m. At 1.5 m, the human birthing level and I/O ratios showed that maximum counts for the total bacteria in autumn were lower indoors than outdoors during the fatting process (I/O values close to one or more bacterial counts indicated higher bacterial contamination inside the building than outside). Furthermore, I/O ratios showed that maximum counts for E. coli, Salmonella, and Aspergillus during autumn were lower indoors than outdoors during the fattening process. All the target houses had indoor sources of airborne bacteria, which contaminated the indoor air. Te outdoor air contamination level was assessed according to Polish Norm standards (Table 4). In the frst week of the chicken fattening cycle at Journal of Environmental and Public Health 1.5 m, the total bacterial counts were 2000 CFU/m 3 , which means that the houses were polluted with a medium extent of pollution. At 0.5 m, the counts from the frst week to the third week were high: 1230 CFU/m 3 , 1438 CFU/m 3 , and 1131 CFU/m 3 , respectively. Biochemical analysis of the isolated strains in the third week of the fattening cycle confrmed the presence of E. coli and Salmonella species (S. enterica and S. subterranean). However, avian pathogenic E. coli (APEC) was not detected. Aspergillus fumigatus was detected by microscopic methods.

Microbial Diversity in Chicken Fattening
Houses. Te analysis of bacterial diversity at the genus and species levels at the end of the fattening cycle-pooled DNA is shown in Figures 9 and 10, respectively. Te tag number of each taxonomic rank (genus, species) in diferent samples was summarized in a profling histogram. Figures 9 and 10 show the taxonomic composition distribution histograms of indoor and outdoor samples at the genus and species levels,    Journal of Environmental and Public Health respectively. Te species with less than 0.5% abundance in all samples were classifed into "others" in other ranks.

Discussion
Counting and monitoring aerial microorganisms inside and outside poultry farms are essential for evaluating the impact of poultry houses on environmental microbiological pollution [24]. Collecting temporal information on the quantity and composition of bioaerosols is necessary to better understand the relationship between these factors and adverse health symptoms in workers and animals. Te numbers and types of airborne bacteria are useful indicators for assessing the adverse efects of human exposure to these emissions [25]. Tis human health risk assessment is used to assess health hazards associated with exposure to airborne bacteria and fungi [26]. Te concentrations and emissions of pathogenic bacteria and fungi in the air depend on the health of the chickens when they are raised in the poultry house.
In this study, the total bacterial population measured in the indoor air and outdoors exhibited a linear relationship during the fattening cycle, with an R 2 of 0.8349. Pyrosequencing analysis of the indoor and outdoor air in chicken fattening houses revealed a large diversity of bacteria in indoor and outdoor air. During the last week of the fattening cycle, a comprehensive DNA analysis was performed on air samples collected from (inside) and (surrounding) poultry houses to identify pathogenic bacteria present during chicken collection for transport to slaughterhouses. Te analysis of indoor air samples demonstrated that Grampositive bacterial species, such as C. kroppenstedtii, S. anginosus, and S. infantis, and Gram-negative bacterial species, such as H. parainfuenzae and Veillonella parvula, are highly abundant in poultry house air during autumn. E. coli was detected in less than 0.02%, whereas Salmonella spp. was not detected; however, Salmonella spp. was detected by culture methods in the third week of the process by biochemical analysis. Te inability to detect Salmonella spp. is in agreement with a previous study [27] that found that Salmonella spp. prevalence in poultry farms is sporadic [28]. Te detected bacterial species belonging to the genera Corynebacterium, Haemophilus, Streptococcus, and Veillonella were potentially harmful to humans [29][30][31][32][33][34][35][36][37][38][39]. Moreover, the detected Aspergillus species are also potentially harmful to humans and broilers [40][41][42]. Tese pathogenic bacteria and fungi were present in a higher percentage inside the house's air than in the air outside, refecting the emission of these microorganisms from the inside air. Although the calculated 1/O refected no pollution, the pyrosequencing analysis of the indoor and outdoor air showed the emission of harmful bacteria to the outdoor environment. Te discharge of potentially pathogenic bacteria from chicken housing to the outdoor environment can pose a considerable health risk to humans and environmental contamination.

Conclusion
Poultry houses are a source of signifcant emissions of microbial pollutants into the atmospheric air. Large discharges of this potentially pathogenic microorganism into the outdoor environment via aerosols from poultry breeding facilities may pose a considerable risk to human health and environmental microbial contamination. To date, there are no reliable data on the relationship between indoor and outdoor microbial contamination in poultry houses in Kuwait. Tis study highlights that poultry houses have the potential to transmit diseases through airborne bioaerosols; therefore, corrective actions are needed to mitigate negative public health impacts. Consideration should be given to establishing an appropriate monitoring system to reduce the types and concentrations of bioaerosols in the air and to take measures to control microbial contamination in the air.

Data Availability
Te original contributions presented in the study are included within the article, and further inquiries can be directed to the corresponding author.

Conflicts of Interest
Te authors declare that they have no conficts of interest.